Novel Compound Hexadecaphlorethol Isolated From Ishige Okamurae And Use Thereof

ABSTRACT

Disclosed herein are hexadecaphlorethol (HdP), which is a novel compound isolated from  Ishige okamurae , and the use thereof in anti-diabetic treatment and exercise performance improvement. The novel compound HdP has the activity of inhibiting α-glucosidase, inducing glucose uptake into differentiated myoblast C2C12 cells, reducing blood glucose levels in zebrafish which have reduced insulin secretion through treatment with alloxan, increasing an intracellular level of calcium ions necessary for muscle contraction and exercise performance in zebrafish, and inducing uptake of glucose, an energy source necessary for muscle contraction, into cells.

FIELD

The present disclosure relates to hexadecaphlorethol, which is a novelcompound isolated from Ishige okamurae, and use thereof and, moreparticularly, to hexadecaphlorethol and the use thereof in anti-diabetestreatment and exercise performance improvement.

BACKGROUND

Diabetes mellitus, commonly referred to as diabetes, is generallyclassified into insulin-dependent diabetes (type I diabetes) andnon-insulin-dependent diabetes (type II diabetes). Insulin-dependentdiabetes is characterized by insulin deficiency resulting from the lossof the insulin-producing beta cells of the pancreatic islets, caused by,for example, viral infection. Non-insulin-independent diabetes istraditionally termed “juvenile diabetes” because a majority of thesediabetes cases occur in young people in their teens or twenties. Asknown by the name itself, insulin-dependent diabetes implies therequirement of external insulin supply for life maintenance.Non-insulin-independent diabetes begins with insulin resistance, acondition in which cells fail to respond to insulin properly, which ismainly caused by obesity. As the disease progresses, insulin, althoughsecreted from the beta cells of the pancreas, may become deficient.Non-insulin-dependent diabetes is mostly found in people in theirthirties or later and is referred to as adult-onset diabetes. The namenon-insulin-dependent diabetes implies that external insulin supply isnot indispensable for life maintenance, but does not mean that insulinis unnecessary for regulating high blood sugar.

Diabetes is accompanied by complications including myocardialinfarction, angina pectoris, damage to the eye, foot ulcers, etc. aschronic high blood sugar lasts with deficient insulin action. Insulinregulates the metabolism of carbohydrates, fats, and proteins bypromoting the absorption of glucose from blood into liver, fat, andskeletal muscle cells. Diabetes is fundamentally an aberrantcarbohydrate metabolism which results, however, in aberrance in proteinand protein metabolism and electrolyte metabolism in vivo. In thiscontext, diabetes is a group of metabolic disorders (J Korean Soc FoodSci Nutr. 2002 31:1071-1077). Therefore, the regulation of blood sugaris believed to be a therapy for preventing or delaying the onset ofacute complications attributed to diabetes.

Carbohydrates in the digestive organs are degraded by α-amylase andα-glucosidase and other digestive enzymes finally into glucose andfructose which are then absorbed into villi of the small intestine. In apatient with diabetes who cannot effectively regulate blood sugar,glucose is not properly processed, thus aggravating the hyperglycemicstate. Inhibition of α-amylase and α-glucosidase in the digestive organsmakes the uptake of glucose slow, thereby regulating post-meal bloodsugar (Int J Obes. 1984. 8 Suppl 1:181-90; Asia Pac J Clin Nutr. 2004.13(4):401-8; J Int Med Res. 1998 October-November 26(5):219-32; DiabetesCare. 1999 Jun. 22(6):960-4).

Particularly, α-glucosidase is an enzyme that acts at the final step ofcarbohydrate digestion to catalyze conversion into glucose. Anα-glucosidase inhibitor can aid to regulate blood sugar by interferingwith the degradation of polysaccharides into monosaccharides to delaysugar uptake (Journal of Life Science. 2008. 18(7):1005-1010; J.Ethnopharmacol. 2000. 72:129-133). According to the mechanism thereof,an α-glucosidase inhibitor interferes with sugar uptake in the smallintestine without acting on the secretion of insulin, thereby minimizingside-effects of conventional drugs, such as hypoglycemia,hepatotoxicity, beta cell hypofunction, etc. (European Journal ofPharmacology. 2009. 615:252-256; Journal of Life Science. 2008.18(7):1005-1010; Food Chem. 2008. 108:965-972).

Widely used as α-glucosidase inhibitors in clinics are acarbose andvoglibose. Particularly, acarbose is known to have a usefulanti-diabetic effect by suppressing glucose uptake in the smallintestine (Eur J Clin Invest. 1994 August, 24 Suppl 3:3-10), but mayproduce gastroenteric troubles such as abdominal distension, abdominalpain, vomiting, diarrhea, etc. as well as side effects, such as gas,diarrhea, and constipation, caused by the degradation of undigesteddisaccharides by bacteria in the large intestine (World J Gastroenterol.2008 Oct. 21. 14(39):6087-92). In contrast, complete inhibition ofα-glucosidase remarkably reduces glucose uptake, causing hypoglycemiaand the use of α-glucosidase initiators is thus followed by problems(Mol Cell Biochem. 1998 May, 182(1-2):101-8). Therefore, there is stillcontinuation of a need for the development of a novel α-glucosidase thatcan reduce such side effects and exert the effect of suppressingpost-meal blood sugar elevation.

Skeletal muscle has the principal role of relaxing and contractingmuscles. In this regard, calcium ions in the cytoplasm of skeletalmuscle cells act as a core secondary transmitter in the contraction andrelaxation of skeletal muscle. Calcium ions play an important extra- andintracellular role in the human body and are minutely controlled tomaintain proper functions thereof in various tissues. Extracellularcalcium is involved in the excitation-contraction coupling of the heartand muscle and in the synaptic transmission of the nerve system as wellas being a main component of the cartilage and bone. Intracellularcalcium is maintained at a concentration 10,000-fold lower than that ofextracellular calcium, playing a key role as a signal transmitter incell division, muscular contraction, cell migration, membranetransmission, and secretion (Clin J Am Soc Nephrol. 2010. 5 Suppl1:S23-30; Nephro Physiol. 2011. 118:22-7; Nat. Rev. Mol. Cell. Biol.2003.4:517-29).

The concentration of intracellular free calcium ions is much lower thanthat of extracellular fluid upon stability, but the depolarization of amyocyte membrane in response to nerve stimulation causes the influx orrelease of calcium ions into the cytoplasm from extracellular fluid oran intracellular calcium reservoir. When the intracellular calciumconcentration reaches a certain level, muscle contraction occurs.Particularly, skeletal muscle contraction persists even upon removal ofcalcium outside myocytes. Hence, muscles are known as an importantintracellular calcium reservoir (Annul. Rev. Physiol. 1976. 38:293-313;Physio. Rev. 1977. 71-108).

Also, an elevated intracellular calcium level is known to inducemitochondrial respiration and ATP production (Am J Physiol Cell Physiol.2004. 287:817-833; Biochemistry. 2013. 52:2793-2809). When muscles aregiven a stimulus, SR (sarcoplasmic reticulum) Ca2+ pumps (SERCA, acalcium pump present on the surface of the sarcoplasmic reticulum movescalcium ions from the cytosol of the cell to the lumen of the SR tocontrol the homeostasis of intracellular calcium ion concentrations) actto transfer calcium ions to the SR to form actin-myosin bonds, resultingin muscle contraction. After muscle contraction, calcium ions returnback to the original position (extracellular space through SR) by activetransport at an expense of ATP which binds to myosin to cleave theactin-myosin bond, allowing muscle relaxation.

The ATP necessary for the active transport is generated through threemetabolic energy pathways including the phosphagen system, glycolysis,and the aerobic system. Particularly, an increase in glucagon release ismost important for producing needed glucose during exercise (J ClinInvest. 1984. 74(4):1404-13). It is reported that exercise increases thenumber of glucose transporter type 4 (GLUT4) expressed in cell membranesand the concentration of GLUT4 in cell tissues (JAMA. 1991. 266:1535-42;J Clin invest. 1979. 64:1011-15; Am J Physiol. 1987. 252:E170-175),which supports the opinion that an increase in the glucose uptake ofmuscles during exercise promotes the regeneration of ATP, increasing therate of glucose transport.

In addition, glycogen depletion in the muscle and the liver duringexercise is a main cause of fatigue and ATP in skeletal muscles is animmediately usable energy source generated in mitochondria upon muscularcontraction. Therefore, a glycogen content in skeletal muscles or an ATPcontent in muscles, measured by using cell experiments or liver ormuscle tissues of experimental animals, is used as a biomarker in thefunctionality evaluation for exercise performance capacity (Guideline offunctionality evaluation for health functional food, 2016, the KoreanMinistry of Food and Drug Safety).

The present disclosure discloses the novel material HdP isolated andidentified from Ishige okamurae and its anti-diabetic activity andactivity of enhancing exercise performance.

SUMMARY

A purpose of the present disclosure is to provide a composition forimproving exercise performance, using HdP, a novel material isolated andidentified from Ishige okamurae.

Other or concrete purposes of the present disclosure will be proposed,below.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and advantages of the presentinvention will be more clearly understood from the following detaileddescription taken in conjunction with the accompanying drawings, inwhich:

FIG. 1 schematically illustrate an HdP isolation process;

FIG. 2 is an HPLC chromatogram of HdP;

FIG. 3 is 1H and 13C NMR spectral data of HdP;

FIGS. 4a and 4b shows two-dimensional NMR spectral HMQC (a) and HMBC (b)data of HdP;

FIG. 5 is an LC-MS-MS spectral data of HdP;

FIG. 6 shows results indicating that HdP induces glucose uptake intodifferentiated myoblast C2C12 cells;

FIG. 7 shows toxicity test results of HdP in zebrafish embryos;

FIG. 8 shows a result illustrating that HdP reduces a blood glucoselevel in adult zebrafishes;

FIG. 9 shows a result illustrating that HdP increases an intracellularcalcium ion (Ca2+) level in zebrafishes; and

FIG. 10 shows a result illustrating that HdP induces glucose influx intozebrafish myocytes.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

As illustrated in the following Examples and Experimental Examples, thenovel material HdP of the following Chemical Formula 1, isolated andidentified from Ishige okamurae, was found to inhibit the activity ofα-glucosidase and to induce glucose uptake in C2C12, which is adifferentiated myoblast cell line. As shown in zebrafish experiments,the novel material was observed to reduce blood glucose levels inzebrafish which was rendered to secrete a reduced level of insulin bytreatment with alloxan, increase an intracellular concentration ofcalcium ions necessary for muscle contraction and motility enhancement,and induce intracellular uptake of glucose, which is an energy sourcenecessary for muscle contraction.

With the above-mentioned experimental results taken into consideration,one aspect of the present disclosure contemplates the compoundrepresented by Chemical Formula 1, a prodrug thereof, a hydrate thereof,or a solvate thereof. Another aspect of the present disclosurecontemplates an anti-diabetic composition comprising the compound ofChemical Formula 1, a prodrug thereof, a hydrate thereof, or a solvatethereof as an effective ingredient. A further aspect of the presentdisclosure contemplates a composition comprising the compound ofChemical Formula 1, a prodrug thereof, a hydrate thereof, or a solvatethereof as an effective ingredient for improving exercise performance.

As used herein, the term “prodrug” refers to a drug that has aphysically or chemically controlled property by chemically modifying atarget drug and is itself biologically inactive, but is converted to abioactive drug by chemical or enzymatic action in vivo afteradministration.

As used herein, “hydrate” means a compound that is associated withwater, without chemical bonds between water and the compound.

As used herein, “solvate” means a compound formed between molecules orions of a solute and molecules or ions of a solvent.

The term “anti-diabetic” or “anti-diabetes”, as used herein, meansalleviation, prevention, or treatment of a symptom of diabetes.

The term “diabetes”, as used herein, means insulin-dependent diabetesmellitus (type 1 diabetes mellitus) and non-insulin dependent diabetesmellitus (type 2 diabetes mellitus) and is further intended to encompassdiabetes resulting from pancreatic damage caused by a differentdisorder, for example, diabetes caused by hyperthyroidism,hyperadrenocorticism, hypersecretion of growth hormone, orhypersecretion of catecholamine, and gestational diabetes.

The term “effective ingredient”, as used herein, means an ingredientthat can exhibit a desired activity by itself or in combination with acarrier which is itself not active.

So long as the effective ingredient exhibits an effect of improvingexercise performance, a certain amount (effective amount) thereof may becontained in the composition of the present disclosure and may vary,depending on use, formulation, etc. Typically, the effective ingredientmay be determined within the range of 0.001% by weight to 15% by weight.Here, the term “effective amount” refers to an amount of an effectiveingredient contained in the composition of the present disclosure whichis capable of producing a desirable medical and pharmaceutical effectsuch as exercise performance improvement, etc., when the composition isadministered to a subject, such as a mammal, particularly a human, for aperiod of administration according to the suggestion of a medicalexpert. Such an effective amount may be empirically determined withinthe typical ability of a person skilled in the art.

In addition to the effective ingredient, the composition of the presentdisclosure may further comprise any compound or natural extract that hasbeen verified for safety and known to have corresponding activities inorder to synergistically reinforce an anti-diabetic effect or anexercise performance improvement effect or to increase the convenienceof administration or intake through addition of similar activities suchas body fat reduction, fatigue alleviation, etc.

Such compounds or extracts may be compounds or extracts described in theofficial compendium of each country, such as pharmacopoeia (e.g., KoreanPharmacopoeia in Korea), health functional food standards codex (e.g.,“Health Function Food Standards and Specifications” notified by theKorean Ministry of Food and Drug Safety in Korea), compounds or extractsapproved according to instructions of each country which regulateproduction and sales of drug products (“the Pharmaceutical Affairs Act”in Korea), and compounds or extracts which are approved forfunctionality according to instructions in each country which regulateproduction and sales of health functional foods (e.g., “the HealthFunctional Foods Act” in Korea). For example, maca gelatinized powder,creatine, Hovenia dulcis pedicle extract powder, and a vegetable wormfermentation extract; a fermented amino acid complex, Hovenia dulcispedicle extract powder, and a Rhodiola sachalinensis extract; andL-arabinose, a nopal extract, cinnamon extract powder, a guava leafextract, digestion-resistant maltodextrin, lyophilized silkworm powder,a Dioscorea batatas alcohol extract, a banana leaf extract, and amulberry leaf extract, which are recognized as having respectivefunctions of “exercise performance improvement”, “fatigue alleviation”,and “blood sugar control”, according to the Health Functional Foods Actin Korea, may fall within the scope of such compounds or extracts.

At least one of such compounds or natural extracts may be contained incombination with the effective ingredient in the composition of thepresent invention.

According to a concrete embodiment, the composition of the presentdisclosure may be regarded as a food.

The food composition of the present disclosure may be prepared into anyform, for example, beverages such as tea, juice, a carbonated beverage,an ion beverage, etc.; processed milk products, such as milk, yogurt,etc.; foods, such as gum, rice cake, traditional Korean sweets andcookies, breads, confectionery, noodles, etc.; and agents for healthfunctional foods, such as tablets, capsules, pills, granules, liquids,powders, flakes, pastes, syrups, gels, jellies, bars, etc. In addition,the food composition of the present invention may be in the form of anyproduct complying with legal and functional classifications according toan enforcement at the time of production and distribution. For example,the composition may be a health functional food according to “the HealthFunctional Foods Act” of Korea or may be confectionery, beans, teas,beverages, or special purpose foods according to the food typesstipulated by the Korean Food Standards Codex (“Health Function FoodStandards and Specifications” notified by the Korean Ministry of Foodand Drug Safety in Korea) of the Food Sanitation Act in Korea.

The food composition of the present disclosure may include a foodadditive in addition to the effective ingredient. A food additive may beunderstood as a substance that can be added to, mixed with, orprecipitated in a food during food preparation, processing, orpreservation. A food additive should be guaranteed safety because it maybe ingested together with food every day and for a long period of time.Safety-guaranteed, food additives are limitedly stipulated in terms ofcomponents or functions by a food additives codex according to an act ineach country (“Food Sanitation Act” in Korea) which regulates theproduction and distribution of foods. The Korean Food Additives Codex(“Food Additive Standards and Specifications” published by the Koreanministry of Food and Drug Safety) stipulates food additives in terms ofcomponents into categories of chemical synthetic products, naturaladditives, and mixed agents. Such food additives may be divided intosweeteners, flavoring agents, preservatives, emulsifiers, acidifiers,thickeners, etc. in view of functions.

A sweetener, which is to provide a food with a sweet taste, may be usedin the food composition of the present invention irrespective of whethernatural or synthetic. Preferable is a natural sweetener. Examples of thenatural sweetener include sugar sweeteners, such as a corn syrup solid,honey, sucrose, fructose, lactose, maltose, etc.

A flavoring agent, which is to make a taste or a smell better, may beused irrespective of whether natural or synthetic. Preferable is anatural flavoring agent. A natural flavoring agent may be used fornutrition as well as enhancing flavor. Natural flavoring agents may beobtained from apples, lemons, tangerine, grapes, strawberries, peaches,etc. or from green tea leaves, Solomon's seals, bamboo leaves, cinnamon,chrysanthemum, jasmine, etc. In addition, a natural flavoring agentobtained from ginseng (red ginseng), bamboo sprouts, aloe vera, ginkgonuts, etc. may be used. The natural flavoring agent may be aconcentrated liquid or an extract of a solid phase. According tocircumstances, a synthetic flavoring agent, such as ester, alcohol,aldehyde, terpene, etc. may be used.

An available preservative may be exemplified by calcium sorbate, sodiumsorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassiumbenzoate, and EDTA (ethylenediaminetetraacetic acid). As an emulsifier,acacia gum, carboxymethylcellulose, xanthan gum, pectin, etc. may beused. Examples of acidifiers include citric acid, malic acid, fumaricacid, adipic acid, phosphoric acid, gluconic acid, tartaric acid,ascorbic acid, acetic acid. An acidifier may be added to adjust a pH ofthe food composition with the aim of restraining microbial growth aswell as improving a taste. Available for use as a thickener are asuspending agent, a precipitating agent, a gelling agent, and a swellingagent.

In addition to the above-mentioned food additives, the food compositionof the present disclosure may further comprise bioactive substances orminerals which are known in the art and guaranteed safety as foodadditives in order to supplement functionality and nutrition.

Such bioactive substances include catechins such as those contained ingreen tea; vitamins such as vitamin B1, vitamin C, vitamin E, vitaminB12, etc.; tocopherol; dibenzoyl thiamine, etc. Examples of availableminerals include calcium agents such as calcium citrate; magnesiumagents such as magnesium stearate; iron agents such as iron citrate;chrome chloride; potassium iodide; selenium; germanium; vanadium; andzinc.

The food composition of the present disclosure may contain theabove-mentioned food additives in respective appropriate amounts enoughto achieve the purposes of addition according to types of the product.

With regard to other food additives available for the food compositionof the present disclosure, reference may be made to a food codex or foodadditive codex according to instruction of each country.

In another embodiment, the composition of the present disclosure may beunderstood as a pharmaceutical composition.

The pharmaceutical composition of the present disclosure may comprise apharmaceutically acceptable carrier in addition to the effectiveingredient and may be prepared into an oral or parenteral formulationaccording to administration routes in a typical manner. Here, the term“pharmaceutically acceptable” means retaining no more toxicity than istoo high for an application (treatment) subject to adapt withoutlimiting the activity of the effective ingredient.

For oral formulations, the pharmaceutical composition of the presentdisclosure may be prepared, together with a suitable carrier, into adosage form, such as a powder, a granule, a tablet, a pill, asugar-coated pill, a capsule, a liquid, a gel, a syrup, a suspension, awafer, etc., according to a method known in the art. In this context,examples of pharmaceutically acceptable carriers include: sugars, suchas lactose, glucose, sucrose, dextrose, sorbitol, mannitol, xylitol;starches, such as corn starch, potato starch, wheat starch, etc.;celluloses such as cellulose, methyl cellulose, ethyl cellulose, sodiumcarboxymethyl cellulose, hydroxypropylmethyl cellulose, etc.; polyvinylpyrrolidone; water; methylhydroxy benzoate; propylhydroxy benzoate;magnesium stearate; mineral oil; malt; gelatin; talc; polyol; andvegetable oil. If necessary, the composition may be formulated, togetherwith a diluent and/or an excipient, such as a filler, a thickener, abinder, a humectant, a disintegrant, etc.

For parenteral formulations, the pharmaceutical composition of thepresent disclosure may be prepared, together with a suitable carrier,into a dosage form, such as an eye drop, an injection, a transdermalagent, a nasal inhaler, a suppository, etc. A carrier suitable for usein an eye drop may be exemplified by an isotonic solution such assterile water, a saline, and 5% dextrose. Optionally, benzalkoniumchloride, methyl paraben, ethyl paraben, etc. may be used forpreservation. When prepared into an injection, the composition mayinclude a carrier such as sterile water, ethanol, polyol, e.g., glycerolor propylene glycol, or a combination thereof. Preferably, a Ringer'ssolution, triethanol amine-containing phosphate buffered saline (PBS) orsterile water for injection, an isotonic solution such as 5% dextrose,etc. may be used. A transdermal agent may be in the form of an ointment,a cream, a lotion, a gel, an external use liquid, a paste, a liniment,an aerosol, etc. A nasal inhaler may be prepared into an aerosol sprayin which a suitable propellant such as dichlorofluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, etc.may be used. For a suppository, Witepsol, Tween 61, polyethyleneglycols, cacao butter, laurin butter, polyoxyethylene sorbitan fattyacid esters, polyoxyethylene stearates, sorbitan fatty acid esters, etc.may be used as a base.

Concrete formulations of pharmaceutical compositions are known in theart. For example, reference may be made to document [Remington'sPharmaceutical Sciences (19th Ed., 1995)], which is incorporated hereinby reference.

The preferable dose of the pharmaceutical composition in accordance withthe present disclosure may vary, depending on various factors includingthe patient's heath state, weight, sex, and age, the severity ofdisease, and the route of administration. For a preferred effect, theeffective amount of the pharmaceutical composition of the presentinvention may range in a daily dose from 0.001 mg/kg to 10 g/kg, andmore preferably from 0.001 mg/kg to 1 g/kg. The composition may beadministered in a single dose, or may be divided into multiple doses perday. Such doses should be construed to limit the scope of the presentdisclosure in no way.

A better understanding of the present invention may be obtained throughthe following examples which are set forth to illustrate, but are not tobe construed as the limit of the present invention.

EXAMPLE

Isolation and Identification of HdP from Ishige okamurae Extract

Example 1: Isolation of HdP

Hdp was isolated from an Ishige okamurae extract as follows, and aschematic view of the isolation process thereof is depicted in FIG. 1.

Ishige okamurae was gathered in an area of Sungsan, Jeju Island, Korea,desalted by washing with fresh water twice, and naturally dried. Thedried Ishige okamurae was ground into powder in a blender and 100 g ofthe powder was immersed in 2 L of 50% ethanol for 24 hours at roomtemperature. Following filtration, concentration at a reduced pressureremoved the solvent to leave an extract in a powder phase.

In 5 L of distilled water was suspended 10 g of the extract powder towhich 5 L of ethyl acetate was then added to give fractions. Removal ofthe solvent by concentration at room temperature afforded an ethylacetate fraction.

For use in centrifugal partition chromatography (CPC), a solvent systemconsisting of n-hexane-ethylacetate-methanol-water was preferentiallyselected through a preparative experiment. Investigation was made intoan optimal partition coefficient K while component ratios in the solventsystem were changed. As a result, a K value of 0.5 was identified to beoptimal when n-hexane, ethylacetate, methanol, and water in the solventsystem were present at a volume ratio of 1:9:4.5:6.5.

The solvent system was put in a separatory funnel and thoroughlyequilibrated by being vigorously shaken at room temperature to separatean upper organic layer and a lower phase which were used as a stationaryphase and a mobile phase, respectively.

In order to perform CPC, the CPC column was filled with the upperorganic phase (stationary phase). While the column was then rotated at1,000 rpm, the lower phase was injected at a flow rate of 2 ml/min tothe column until the pump pressure was constantly maintained. After thepressure reached a constant value, 60 ml of a sample (a solution of 500mg of the ethylacetate fraction in 6 mL of a mixture of 1:1 of water andmethanol) was injected. While the effluent was monitored at 230 nm,fractions were collected. Relatively non-polar fractions were separatedat 70 min to 120 min after the sample injection.

Then, the CPC fractions thus obtained were subjected to HPLC, using asemi-preparative HPLC column (YMC-Pack ODS-A, 10×250 mm, 5 μm particlesize, YMC Co Ltd, Tokyo, Japan). In brief, 32% acetonitrile containing0.1% formic acid and 68% water containing 0.1% formic acid were used asa mobile phase which was allowed to flow at a flow rate of 2 mL/min. Theeffluent was monitored with a UV detector at 230 nm and a compoundhaving the second highest intensity, detected at 17.302 min, wasisolated. An HPLC chromatogram accounting for the compound is depictedin FIG. 2.

Example 2: Identification of the Compound

For structural identification of the isolated compound, 1H NMR, and 13CNMR spectra were measured. HMQC and HMBC spectra, which aretwo-dimensional (2D) NMR spectra, were measured to analyze correlationbetween hydrogen atoms themselves and between hydrogen and carbon atoms.The molecular weight was determined using LC-MS-MS. The results areshown in FIGS. 3 to 5, respectively. Comparison of the data with thosein related documents concluded the identification of HdP, a novelcompound represented by Chemical Formula 1.

Experiment Examples: Assay of HdP for Anti-Diabetic Activity andExercise Performance Improvement Experiment Example 1: Anti-DiabeticActivity of Hdp

1.1 Assay of Inhibitory Activity Against α-Glucosidase

Inhibitory activity against α-glucosidase was evaluated using a yeastenzyme according to the method of Watanabe et al. (Watanabe, Kawabata,Kurihara, & Niki, 1997). In the reaction condition of pH 7.0 and 37° C.,5 mM p-nitrophenyl α-D-glucopyranoside (PNP-G) serving as a substratewas reacted with a dilution of 32 mU/ml enzyme (yeast α-glucosidase,Sigma) in 100 mM phosphate buffer and inhibitory activity was measuredusing spectrophotometry. The sample was reacted with PNP-G for 5 min andthe absorbance of 4-nitrophenol released upon the hydrolysis of PNP-Gwas read at 405 nm. Subsequently, the same amount of the substratesolution was added and reacted at room temperature for an additionalfive min., after which the reaction was stopped by adding 0.5 M Na₂CO₃.A change in absorbance at 405 nm was measured.

Inhibitory activity (%) against α-glucosidase was calculated accordingto the formula (1-absorbance of sample-added group/absorbance ofsolvent-added group)×100).

Acabose, which is an oral hypoglycemic agent inhibitory ofα-glucosidase, was used as a positive control.

1.2 Measurement of Glucose Uptake of C2C12 Cell

1.2.1 C2C12 Cell Culture and Differentiation

The mouse-derived myoblast C2C12 cells (ATCC, Manassas, Va., USA) weresuspended in DMEM (Dulbecco's Modified Eagle's Media) mediumsupplemented with 10% FBS and an antibiotic and cultured at 37° C. undera 5% CO₂ atmosphere in an incubator. Upon 80% confluency, the C2C12cells subjected to differentiation in a DMEM medium containing 2% horseserum and a low concentration of glucose for 5 days, with the mediumfreshly changed every three days. The differentiated cells were serumstarved for 12 hours in serum-free DMEM containing a low concentrationof glucose, washed with PBS, and then reacted for 24 hours with thesample (HdP) in a fresh serum-free DMEM.

1.2.2 Measurement of Glucose Uptake of C2C12 Cell Using Flow Cytometry(FACS)

Glucose uptake of mouse-derived myoblast C2C12 cells was measured usingflow cytometry (FACS) according to the (2-(N-(7-nitrobenz-2 oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) assay method (Cell DeathDis. 2017 Oct. 5. 8(10):e3078). The cultured cells were incubated with10 μM 2-NBDG at 37° C. for 24 hours and then analyzed using BD FACSCantoll (BD Biosciences, NJ, USA), with the fluorescence of 2-NBDGdetected in a FITC channel.

1.3 Animal Test

For use in experiments, adult zebrafish were purchased from a market(Jeju Aquarium, Jeju Island, Korea). They were acclimated to a conditionof a temperature of 28.5±1° C. and a 14/10 h light-dark cycle in a 3.5 Lacryl tank and fed twice a day (Tetra GmgH D-49304 Melle, made inGermany). Experiments with zebrafish were approved by the InstitutionalAnimal Care and Use Committee of Jeju University.

1.3.1 Test of HdP Toxicity in Zebrafish Embryo

Four days after fertilization, each of the embryos (n=15) wastransferred to 12-well plates, each containing 950 μl of an embryomedium (distilled water containing 60 ppm salt) and 50 μl of HdP (0.01,0.1, 1, and 10 μg/ml) and the viability of zebrafish embryos exposed toHdP for 168 hours after fertilization were measured.

1.3.2 Measurement of Blood Sugar Level in Zebrafish

Wild-type adult zebrafish were treated for 1 hour with 2 mg/ml alloxanand then for 1 hour with 1% glucose in the absence of insulin.Subsequently, the reaction solution was exchanged with water in whichthe zebrafish were then left for 1 hour before treatment with HdP,metformin, or BAPTA-AM for 90 min. Thus, the experiment groups includeda non-treated group, an alloxan-treated group (control), a group treatedwith alloxan and HdP (0.3 μg/g body weight), a group treated withalloxan and metformin (5 μg/g body weight), a group treated with alloxanand BAPTA-AM (3 μg/g body weight), and a group treated with alloxan,BAPTA-AM(3 μg/g body weight), and HdP (0.3 μg/g body weight).

2. Test Results

2.1 Measurement Result of Inhibitory Activity Against α-Glucosidase

Concentrations of the samples which were necessary for inhibiting theactivity of α-glucosidase by 50% (IC₅₀) are given in Table 1, below.

TABLE 1 Sample IC₅₀ (μM) Hexadecaphlorethol(HdP) 54.97 Acarbose 1050.23

As is understood from the data of Table 1, HdP according to the presentdisclosure exhibited higher inhibitory activity against α-glucosidasethan acarbose did.

2.2 Measurement Result of Glucose Uptake of C2C12 Cell

Glucose uptake by HdP in differentiated CeC12 cells was measured and theresults are depicted in FIG. 7. FACS is an instrument isolatingfluorescence-marked cells from a heterogeneous cell mixture. In C2C12cells which were treated with the fluorescence-marked glucose analogue2-NBDG, fluorescence intensity became stronger with the detection ofmore 2-NBDG through a FITC filter. Thus, the detected cell populationsmigrated right in the graph. With reference to FIG. 6, glucose uptakeinto the differentiated C2C12 cells treated with HdP for 24 hours isshown to significantly increase in a dependent manner on the dose ofHdP, indicating that HdP can lower a blood glucose level. Glucose uptakeinto differentiated C2C12 cells is known as an index accounting for areduction in blood glucose level (Diabetes 30 (1981) 1000-1007;Biochemical and Biophysical Research Communications 420 (2012) 576-581;Nat. Rev. Mol. Cell Biol. 7 (2006) 85-96; Eur. J. Cell Biol. 87 (2008)337-351)

2.3 Test Result of HdP Toxicity in Zebrafish Embryo

In order to examine a toxicity-free concentration of HdP at which notoxicity occurs, survival rates of zebrafish embryos were evaluated. Asshown in FIG. 7, the embryos were observed to survive 1 μg/ml or lessHdP at a rate of about 90% or higher.

2.4 Measurement Result of Blood Glucose Level of Zebrafish Under Controlof HdP

Examination was made to see whether HdP controls blood sugar in ananimal model. In this regard, adult zebrafish which had been treatedwith alloxan to release a reduced level of insulin were injected withglucose and monitored for blood glucose levels.

Results are depicted in FIG. 8. With reference to FIG. 8, HdP wasobserved to significantly reduce blood glucose levels of alloxan-treatedadult zebrafishes in a dose-dependent manner. This effect was similar tothat of metformin, which is commercially used to control blood glucose.Particularly, when account is taken of the fact that HdP was used at aconcentration of 0.3 μg/g in contrast to 5 μg/g metformin, a similarglucose uptake effect is expected even with a concentration of HdP whichis 16.7-fold lower than that of metformin.

Experiment Example 2: Activity of HdP to Improve Exercise Performance

1. Experiment Method

1.1 Preparation of Zebrafish

For use in experiments, adult zebrafish were purchased from a market(Jeju Aquarium, Jeju Island, Korea). They were acclimated to a conditionof a temperature of 28.5±1 C° and a 14/10 h light-dark cycle in a 3.5 Lacryl tank and fed twice a day (Tetra GmgH D-49304 Melle, made inGermany). Experiments with zebrafish were approved by the InstitutionalAnimal Care and Use Committee of Jeju University.

1.2 Measurement of Intracellular Calcium Level in Zebrafish Embryo UsingFluo-4

Intracellular calcium levels of zebrafish embryos were measured using acalcium-sensitive Fluo-4 probe. Embryos were incubated with Fluo-4 at28.5±1 C for 30 min before treatment with HdP (0.6, 3, and 6 μg/ml). Inorder to block the entry of calcium into cells, the embryos were reactedwith the calcium chelator BAPTA-AM 0.1 mM for 1 hour before incubationwith Fluo-4. The experiment groups included a non-treated group (N), agroup treated with Fluo-4 alone (F), a group treated with HdP alone(0.6, 3, and 6 μg/ml), a group treated with Fluo-4 and HdP (0.6, 3, and6 μg/ml), and a group treated with Fluo-4, BAPTA-AM, and HdP (6 μg/ml).

1.3 Measurement of Blood Glucose Level of Zebrafish

Wild-type adult zebrafish were treated for 1 hour with 2 mg/ml alloxanand then for 1 hour with 1% glucose in the absence of insulin.Subsequently, the reaction solution was exchanged with water in whichthe zebrafish were then left for 1 hour before treatment with HdP,metformin, or BAPTA-AM for 90 min. Thus, the experiment groups includeda non-treated group, an alloxan-treated group (control), a group treatedwith alloxan and HdP (0.3 μg/g body weight), a group treated withalloxan and metformin (5 μg/g body weight), a group treated with alloxanand BAPTA-AM (3 μg/g body weight), and a group treated with alloxan,BAPTA-AM (3 μg/g body weight), and HdP (0.3 μg/g body weight).

2. Test Result

2.1 Measurement Result of Intracellular Calcium Level Change by HdP inZebrafish

Intracellular calcium level changes by HdP in zebrafish embryo cellswere observed with Fluo-4. Referring to FIG. 9, a significant differenceappeared around the abdomen between the non-treated group (N) and theFluo-4-treated group (F). The group treated with HdP alone did notundergo a special phosphorescence change, compared to the non-treatedgroup (N) or the Fluo-4-treated group (F) (left panel in FIG. 9),indicating no HdP-induced phosphorescence change. A significant increasein phosphorescence was observed in the group treated with HdP and Fluo-4in combination, compared to the Fluo-4-treated group (F), indicatingthat treatment with HdP increases intracellular Ca²⁺ levels. Aphosphorescence decrease in the group treated with the calcium chelatorBAPTA-AM in combination with HdP identified the intracellular influx ofCa²⁺ (right panel in FIG. 9).

2.2 Measurement Result of Blood Glucose Level Change by HdP in Zebrafish

Examination was made to see whether HdP induces the uptake of glucosenecessary for muscle contraction with the increase of intracellular Ca²⁺levels. In this regard, adult zebrafish which had reduced insulinsecretion by treatment with alloxan were injected with glucose tomonitor HdP-induce glucose influx into myocytes in terms of bloodglucose levels. The results are shown in FIG. 10. A reduced level of theenergy source glucose in blood is known as an index accounting forglucose influx into tissues, such as muscles, etc. (Diabetes 30 (1981)1000-1007; Biochemical and Biophysical Research Communications 420(2012) 576-581; Nat. Rev. Mol. Cell Biol. 7 (2006) 85-96; Eur. J. CellBiol. 87 (2008) 337-351).

Blood glucose levels in the alloxan-treated adult zebrafishes weresignificantly reduced by HdP. This effect was similar to that ofmetformin, which is commercially used to control blood glucose.Particularly, when account is taken of the fact that HdP was used at aconcentration of 0.3 μg/g in contrast to 5 μg/g metformin, a similarglucose uptake effect is expected even with a concentration of HdP whichis 16.7-fold lower than that of metformin.

A relation between an effect of HdP on glucose influx and an increase ofintracellular calcium levels was investigated. For this, zebrafish weretreated with the calcium chelator BAPTA-AM in combination with alloxanor HdP. No significant differences in blood glucose level were observedbetween the groups treated with alloxan plus BAPTA-AM and with HdP plusBAPTA-AM, indicating that glucose uptake in adult zebrafish increaseswith the HdP-induced increase of intracellular calcium levels.

Provided according to the present disclosure is a composition forimproving exercise performance, using a novel compound HdP isolated fromIshige okamurae, as described hitherto. The composition of the presentinvention may be prepared into a food or drug product.

Although the preferred embodiments of the present invention have beendisclosed for illustrative purposes, those skilled in the art willappreciate that various modifications, additions and substitutions arepossible, without departing from the scope and spirit of the inventionas disclosed in the accompanying claims.

What is claimed is:
 1. A compound of Chemical Formula 1, a hydratethereof, or a solvate thereof:


2. The composition comprising the compound of claim 1 and an excipient.3. The composition of claim 2, which is in a form of a food composition.4. The composition of claim 2, which is in a form of a pharmaceuticalcomposition.
 5. A method for improving exercise performance ability, themethod comprising: administering to a patient in need thereof atherapeutically effective amount of the composition of claim
 2. 6. Themethod of claim 5, wherein the composition is in a form of a foodcomposition.
 7. The method of claim 5, wherein the composition is in aform of a pharmaceutical composition.